Airway smooth muscle (ASM) remodeling in chronic asthma involves structural and functional changes from healthy into hyper-reactive and proliferative/hypertrophic ASM. ASM remodeling is an important determinant in airway obstruction and decline pulmonary function in asthma that compounds the well- established immune/inflammatory components. These ASM phenotypic changes are of great clinical importance yet remains poorly understood. A more recent concept is that ASM not only contributes to physical obstruction of airways, but acts as immune effector in asthma. Orai1 encodes canonical, ubiquitous and evolutionarily-conserved Ca2+ release-activated Ca2+ (CRAC) channels regulated by the endoplasmic reticulum (ER) Ca2+ sensor STIM1. We showed that CRAC mediate proliferative signals during pathological smooth muscle remodeling. However, Orai1-mediated CRAC is widely functional in most, if not all, tissues rendering its specific targeting for therapy challenging. Orai1 has two homologs, Orai2 and Orai3 which are exclusive to vertebrates and mammals, respectively. We propose that Orai3 is a key determinant in ASM remodeling during asthma and that Orai3 is a better therapeutic target than Orai1. Our exciting data identified novel native Orai3/Orai1 heteromultimeric channels in ASM that are regulated by STIM1 and gated by cytosolic leukotrieneC4. This intracrine function of leukotrieneC4 is independent of paracrine actions on plasma membrane receptors. We showed upregulation of STIM1, Orai1 and Orai3 proteins in ASM from asthmatic mice. Furthermore, we generated Orai3 knockout (O3-KO) mice which, unlike Orai1 knockouts, are viable and healthy. Therefore, we hypothesize that: i) increased leukotrieneC4-regulated Ca2+ (LRC) currents are regulated by STIM1 and mediated by Orai3/Orai1 hetero-multimers in ASM is an important determinant for ASM remodeling in asthma. We propose two specific aims to address this hypothesis: Aim1) determine the mechanisms of leukotrieneC4-dependent activation of Orai3/Orai1 in ASM. i) We will determine the domains of STIM1 and Orai3 required for Orai3/Orai1 channel activation by LTC4. Using STIM1 truncations, Orai3/Orai1 chimeras and erase and replace strategies coupled to multi-cell Ca2+ imaging, we will determine the exact domains of STIM1 and Orai3 required for STIM1/Orai3 interaction and LTC4 action on Orai3/Orai1 channels. ii) We will determine the signaling pathways controlled by LRC-generated Ca2+ signals downstream Akt activation. Aim 2) We will determine the role of STIM1 and Orai3 in ASM remodeling in a mouse model of asthma. i) We will determine the increase in LRC current activity and Akt signaling in ASM from ovalbumin (OVA)-induced asthmatic mice. ii) We will determine the ability of smooth muscle-specific STIM1 and Orai3 knockout mice to develop asthma and ASM remodeling upon OVA challenge. These studies will enhance our understanding of native Orai Ca2+ channels regulation in ASM and will validate Orai3 and Orai3-encoded channels as therapeutic targets for ASM remodeling in chronic asthma.